Cytoskeletal dynamics regulate a cell?s ability to polarize and to move. This proposal focuses on the regulation of actin dynamics by Ena/VASP proteins. Ena/VASP proteins localize to within cells to actin-rich regions associated with cell motility including the lamellipodia and filopodia. In vivo studies of Ena/VASP proteins in mice and flies reveal cellular defects consistent with a role for Ena/VASP proteins in the regulation of cytoskeletal dynamics, but the precise mechanism of Ena/VASP function is unknown. Clonal cell lines from Mena-/-VASP-/- knock-out mice will be isolated on the basis of their motility properties in tissue culture. Retroviral transformation of these cell lines with novel Mena mutants will permit a high-resolution analysis Ena/VASP function in the context of living cells. Live and fixed cell microscopy, biochemistry, and tissue culture techniques will be used to characterize these cells in functional assays including random cell motility, chemotaxis, and Listeria motility. These efforts will define region(s) of Ena/VASP proteins essential for function in mammalian fibroblastic cells, and provide the basis for a biochemical screen for Mena interactors.